RESUMO
The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Antígeno de Maturação de Linfócitos B/metabolismo , Sistemas Automatizados de Assistência Junto ao Leito , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/patologia , Citocinas/metabolismo , Linfócitos T , CriopreservaçãoRESUMO
Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.
Assuntos
Antígeno de Maturação de Linfócitos B/metabolismo , Citometria de Fluxo , Reação em Cadeia da Polimerase , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Antígeno de Maturação de Linfócitos B/genética , Biomarcadores , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos Quiméricos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologiaRESUMO
Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) Tcells. In order to overcome a lottolot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serumfree media [PrimeXV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ TCell Expansion XenoFree Medium, Takara Bio™ (TB) and TCM GMPPrototype, CellGenix™ (CG)] to the standard CAR Tcell medium containing FBS (RCF). After 12 days of CD19.CAR Tcell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR Tcells was evaluated using intracellular staining, a chromium release assay and a longterm coculture assay. Expansion and viability did not differ between the CAR Tcells generated in serumfree media compared to the standard FBScontaining medium. The CG CAR Tcells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNFα+ CAR Tcells than the CAR Tcells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR Tcells generated with serumfree media exhibited a higher cytotoxicity than the CAR Tcells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR Tcell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyteactivation gene 3 and Tcell immunoglobulin and mucindomain containing3. The CAR Tcells cultured in FF had a higher percentage of central memory CAR Tcells (40.0 vs. 14.3%, P=0.0470) than the CAR Tcells cultured with FBS, whereas the CAR Tcells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR Tcells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serumfree media. Given the advantages of freedom from bovine material and consistent quality, serumfree media hold promise for the future development of the field of GMP manufacturing of CAR Tcells.